1. Field of the Invention
The present invention relates to dyeing agents for the biological stain of red blood cells, white blood cells, epithelial cells or the like. In particular, the present invention relates to dyeing agents for bio samples like urine, containing extracellular tangible components such as crystals or casts; these components are difficult to discern from dye precipitates, urinary components deposited or coagulated, impurities such as dust, etc. which may be contained in the same biological sample, in addition to the aforesaid cellular components. The present invention also relates to a method for preparing such dyeing agents and to the use thereof.
The present invention further relates to an apparatus for particle image analysis in which images of particles suspended in flowing fluids are photographed and analyzed, and in particular, to an apparatus for analysis of particle images to determine the kind of cells or particles in blood or urine, or culture cells, etc. and the life or death of these cells.
2. Description of the Related Art
For classification and analysis of cells in blood or cells and particles in urine which have been employed in the prior art, a specimen is put on a glass slide and observed microscopically. In the case of urine having a low density of particles therein, a urinary sample is previously centrifuged with a centrifuging machine, and the thus-concentrated sample is microscopically observed. These operations for observation and inspection have been automated using an apparatus through the steps of applying the sample onto the glass slide, setting the glass slide in a microscope, automatically scanning a stage of the microscope to stop it at the position where particles are present, taking a picture of still images of the particles, and classifying the particles in the sample using characteristic extraction and pattern recognition techniques by image processing. However, such a process encounters problems in that it takes time for preparing a sample, and furthermore, additional operations are necessary for locating particles while mechanically moving the microscope stage and transferring the particles to an appropriate zone for image input. Thus, the time period for analysis is prolonged and the apparatus becomes complicated.
Flow cytometry for optically analyzing an analyte suspended in a fluid sample while flowing the sample in a flow cell is known. In flow cytometry, it is unnecessary to prepare a smear instead, a fluorescent or scattering intensity of each particle in a sample is determined. A flow cytometer has the ability to process 1000 particles per second. However, it is still difficult to obtain information which reflects morphological characteristics of particles. Flow cytometry is thus unable to classify particles by their morphological characteristics with the effective of microscopic observation.
An attempt has been made to take a photograph of particle images in a continuously flowing sample and analyze and classify the particles from the respective particle images; see Japanese Patent Application KOHYO No. 57-500995 and Japanese Patent Application KOKAI (Laid-Open) No. 63-94156.
Japanese Patent Application KOHYO No. 57-500995 discloses a method for analysis of particles which comprises passing a sample through a path in a special shape, flowing particles in the sample there in a wide zone for photographing, taking a picture of still images by a flash lamp, and analyzing the images. According to the method, the flash lamp, which is a pulse light source, periodically emits light by synchronizing with a CCD camera and, enlarged images of sample particles are projected on the CCD camera using a microscope. The emission time of the pulse light source is short so that still images can be obtained even though particles flow continuously. In addition, 30 sheets/sec of still images can be photographed with a CCD camera.
Japanese Patent Application KOKAI No. 63-94156 discloses a method which comprises providing a particle-detecting optical system other than a still image photographing system, upstream a zone for photographing particle images in a sample flow, previously detecting the passing particles at the particle detection zone, and flashing a lamp with an appropriate timing when the particles reach the zone for photographing particle images. According to this method, the passing particles can be detected without periodic emission of a pulse light source, and still images can be photographed only when the particles reach the photographing zone. Accordingly, particle images can be efficiently obtained. Even when a sample has a low concentration, there is no chance to process meaningless images where no particles are present.
On the other hand, when urinary sediments in urine are analyzed, microscopic observation is conventional without using any staining solution; only where it is difficult to distinguish an analyte is a dyeing agent for urinary sediments employed. For this technique, reference is made to, e.g., "KENSA-TO-GIJUTSU", Igaku Shoin Publishing Co., vol. 10, No. 9 (1982:9), 846-850, and Japanese Patent Application KOKAI No. 5-40118.
As listed in the above publications, conventional dyeing agents for urinary sediments include the Sternheimer dyeing agent, the New Sternheimer dyeing agent, the Sternheimer-Malbin dyeing agent, etc.
The Sternheimer dyeing agent consists of Solution I: 2% aqueous National Fast Blue solution, and Solution II: 1.5% aqueous Pyronin B solution. To prepare the dyeing agent, Solution I was blended with Solution II in a 1:1 ratio.
The New Sternheimer dyeing agent consists of Solution I: 2% aqueous Alcian Blue solution, and Solution II: 1.5% aqueous Pyronin B solution. To prepare the dyeing agent, Solution I was blended with Solution II in a ratio of 2:1.
The Sternheimer-Malbin dyeing agent consists of Solution I obtained by dissolving 3.0 g of Crystal Violet in 20.0 ml of 95% ethanol, adding 0.8 g of ammonium oxalate to the solution, and diluting the mixture with 80.0 ml of purified water; and Solution II obtained by dissolving 0.25 g of Safranine O in 10.0 ml of 95% ethanol and diluting the solution with 100.0 ml of purified water. To prepare the dyeing agent, Solution I was blended with Solution II in a ratio of 3:97.
In flow cytometry in which urinary sediments in continuously flowing urine are photographed and the sediments are analyzed and classified from the respective images of the sediments, improvement in image processing efficiency depends on cutting-out of images to be analyzed, extraction of characteristic parameters such as color, shape, size, etc. It is thus required to stain the analyte for improving the image processing efficiency.
Staining for urinary sediments using the conventional Sternheimer dyeing agent and New Sternheimer dyeing agent is a highly useful method for supravital staining which is excellent in visual recognition of sediment components in urine.
However, these dyeing agents consist of polar molecules in which charges in the constituent dye molecules are localized. As a result, when dyeing agents are mixed with urine, agglutination of proteins, sugars, glycoproteins, etc. dissolved in urine is caused to form the agglutination products. For this reason, when it is attempted to photograph particle images of urinary sediments stained with these dyeing agents, the agglutination products interfere as impurities to seriously reduce the detection efficiency of urinary sediments or cause clotting of the flow path. Furthermore, depending upon the agglutination products, their shape might be misread as casts; or where cells, casts or blood cells are hidden in an agglutination mass, these components might be overlooked.
In addition, flow cytometry involves a problem that hemolysis results in an inaccurate count of red blood cells. Furthermore, the agglutination products which should be discarded are also labeled in a step of image processing so that the speed of classification processing decreases or image memory capacity becomes insufficient.
On the other hand, staining for urinary sediments using the Sternheimer-Malbin dyeing agent is inferior in visual recognition of discernible components in urine to the aforesaid staining. In addition, tyrosine-like needles are sometimes formed and might be misread as crystals derived from urine. Furthermore, the Sternheimer-Malbin dyeing agent involves a serious problem in that the count of red blood cells becomes inaccurate due to hemolysis caused by the dyeing agent, as in the staining described above.
As stated above, the conventional dyeing agents could not provide any satisfactory results in an apparatus for image analysis of flow type stain particles in which an analyte component in a continuously flowing sample is photographed to perform image analysis.